1. Weed Control
The use of selective herbicides for controlling specific weeds or plants in crops has become almost a universal practice. The market for these herbicides approaches a billion dollars annually. Even with this extensive use, weed control remains a significant and costly problem for the farmer.
Present day herbicides used singly or in so-called tank mixes require careful management to be effective. Time and method of application and stage of weed plant development are critical to getting good weed control with herbicides. Application of large amounts of preemergence herbicides can result in a commitment to grow the same crop in subsequent years because of chemical persistence in the soil which prevents rotation with a crop sensitive to that herbicide. Furthermore, some weed species are simply resistant to the available herbicides. Therefore, the development of effective herbicides increases in importance every year, especially as other weeds are controlled and thus reduce interplant competition.
Weed control in maize is currently accomplished by soil application of herbicides that are applied before the crop emerges and prior to the observation of a weed problem. The preemergence herbicides currently used adequately control most dicot and monocot (grass) weeds in maize. However, annual grass weeds such as wild proso millet and wooly cupgrass and perennial grass weeds commonly escape preemergence weed control. Preemergence herbicides require rainfall for activation, and under low rainfall conditions they fail to control grass weeds in corn. Furthermore, some preemergence herbicides persist in the soil and several have been detected as groundwater contaminants. The options for controlling these escape grass weeds are very limited. A postemergence herbicide for grass weed control in maize would be very beneficial. An attractive alternative to developing new herbicides to combat this weed control problem in maize and/or to decrease the amount of herbicide carryover and groundwater contamination in maize fields from the existing herbicides is to develop maize hybrids or varieties that are tolerant to other existing herbicides that normally kill all monocot (grass) species. The herbicide POAST.TM. (BASF Corp., Parsippany, N.J.) kills most grasses, and is applied at lower rates than many preemergence herbicides. POAST.TM. is nonpersistent in the environment and therefore does not represent a groundwater contamination threat. POAST.TM.-tolerant maize would provide the producer with increased weed management flexibility because POAST.TM. could be applied when a grass weed problem was detected without risk of damage to the crop and only to the areas with a weed problem. Therefore, postemergence control of local weed problems would further decrease the amount of herbicide applied compared to existing preemergence weed control strategies.
2. Tissue Culture of Maize
Irrespective of the plant species, there are a number of common features that apply to most tissue culture programs. The technique of cell and tissue culture has been widely developed, and much work has been done on growth, metabolism and differentiation of tissue culture of dicotyledons (Yamada, 1977, in Plant Cell, Tissue and Organ Culture., eds. Reinert and Bajaj, pp. 144-159, Springer-Verlag, Berlin). However, successful tissue culture studies with monocotyledons (e.g., the cereal crops such as maize, rice, wheat, barley, sorghum, oats, rye and millet) leading to plant regeneration are not as widely documented as with dicotyledons. Success is frequently dependent on choosing donor tissues for culture initiation which come from plants of appropriate genotype as well as physiological and development states. Other features which are obviously important include the organic and inorganic composition of the growth medium and the physical environment in which the cultures are grown.
The development of maize tissue cultures capable of plant regeneration was accomplished after the identification of appropriate genotypes and donor tissues (Green and Rhodes, 1982, in Maize for Biological Research, ed. W. F. Sheridan, pp. 367-371, Plant Molecular Biology Association, Charlottesville, Va.). The first method developed which regenerated plants from tissue cultures of maize used immature embryos as donor tissues. Another donor tissue from which regenerable tissue cultures of maize have been initiated are immature tassels. This tissue is the male flower and as it matures, it is responsible for pollen production. Immature embryos, inflorescences, and the few other tissues in cereals from which regenerating cultures have been initiated all have the common characteristic of juvenility. With N6 or Murashige-Skoog (MS) growth media (defined below in Example 3) and a synthetic auxin, such as 2,4-dichlorophenoxyacetic acid (2,4-D), tissue cultures develop rapidly from the scutellum of the embryos. The resulting cultures are developmentally heterogeneous and contain a variety of tissue types. Removal of the 2,4-D from the growth medium permits these cultures to produce large numbers of regenerated plants. Cultures of this type have proved capable of regenerating plants for up to three years.
Regenerated plants obtained from tissue cultures are grown to maturity in a glasshouse, growth chamber, or field. The progeny seed produced from crosses with regenerated plants permits the evaluation of subsequent generations. The basic tissue culture methods developed for corn have been extended to many other cereal species.
An interesting development in recent years has been the occurrence of somatic embryogenesis in tissue cultures of maize. Somatic embryogenesis is the process where cells from callus, suspension, or protoplast cultures develop into complete embryos similar to zygotic embryos produced in seeds. It is now possible to reliably initiate cultures of corn which have two important characteristics. One is that the callus cultures are friable, meaning that they are soft and loose in texture. This property is important because cultures of this type exhibit rapid growth and are facilitated in the initiation of suspension cell cultures. The other valuable attribute of these friable cultures is their ability to form very large numbers of somatic embryos. Microscopic examination reveals the presence of many small, organized structures on the surface of the callus. These structures are young somatic embryos at various developmental stages. These friable cultures will retain their embryogenic potential for as long as two years, and have shown the capacity to produce extremely large numbers of somatic embryos.
The somatic embryos in these friable calli develop to maturity when the cultures are transferred to medium containing an increased concentration of sucrose (e.g., 5-6%) and no hormones. After approximately two weeks of growth on this medium, many embryos have become quite mature. They germinate rapidly and grow into plants when placed on MS or N6 medium containing 2% sucrose. The plants are then established in soil and are grown to maturity.
It is now well-documented that a high level of genetic variability can be recovered from plant tissue culture. It is well documented that spontaneous genetic variability in cultured plant cells may be the result of mutation (Meredith and Carlson, 1982, in Herbicide Resistance in Plants, eds. Lebaron and Gressel, pp. 275-291, John Wiley and Sons, N.Y.). The frequency of mutants can also be increased by the use of chemical or physical mutagens. Some of this variability is of agronomic importance. Mutants for disease resistance have been obtained in sugar cane for Fiji disease, early and late blight in potato, and southern corn leaf blight in maize. In rice, maize, and wheat, considerable variability for traits inherited as single genes of plant breeding interest has been recovered, including time of seed set and maturation, seed color and development, plant height, plant morphology, and fertility.
3. Mechanisms of Herbicide Tolerance
There are three general mechanisms by which plants may be resistant to, or tolerant of, herbicides. These mechanisms include insensitivity at the site of action of the herbicide (usually an enzyme), rapid metabolism (conjugation or degradation) of the herbicide, or poor uptake and translocation of the herbicide. Altering the herbicide site of action from a sensitive to an insensitive form is the preferred method of conferring tolerance on a sensitive plant species. This is because tolerance of this nature is likely to be a dominant trait encoded by a single gene, and is likely to encompass whole families of compounds that share a single site of action, not Just individual chemicals. Therefore, detailed information concerning the biochemical site and mechanism of herbicide action is of great importance and can be applied in two ways. First, the information can be used to develop cell selection strategies for the efficient identification and isolation of appropriate herbicide-tolerant variants Second, it can be used to characterize the variant cell lines and regenerated plants that result from the selections.
4. Herbicide Tolerance Selection
Tissue culture methods have been used to select for resistance (or tolerance) using a variety of herbicides and plant species (see review by Meredith and Carlson, 1982, in Herbicide Resistance in Plants, eds. Lebaron and Gressel, pp. 275-291, John Wiley and Sons, N.Y.). For example, P. C. Anderson et al. in U.S. Pat. No. 4,761,373, disclose the use of tissue culture methods to produce maize plants resistant to herbicidal imidazolinones and sulfonamides. The resistance is due to the presence of altered acetohydroxy acid synthase which is resistant to deactivation by these herbicides.
5 Herbicidal Cyclohexanediones
Certain 1,3-cyclohexanediones exhibit general and selective herbicidal activity against plants. One such cyclohexanedione is sethoxydim {2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1 -one}. Sethoxydim is commercially available from BASF (Parsippany, N.J.) under the designation POAST.TM..
Other herbicidal cyclohexanediones include clethodim, (E,E)-(.+-.)-2-[1-[[(3-chloro-2-propenyl)oxy]imino]propyl]-5-[2-(ethylthio )propyl]-3-hydroxy-2-cyclohexen-1-one; available as SELECT.TM. from Chevron Chemical (Valent) (Fresno, Calif.); cloproxydim, (E,E)-2-[1-[[(3-chloro-2-prophenyl)oxy]imino]butyl]-5-[2-(ethylthio)propyl ]-3-hydroxy-2-cyclohexen-1-one; available as SELECTONE.TM. from Chevron Chemical (Valent) (Fresno, Calif.); and tralkoxydim, 2-[1-(ethoxyimino)propyl]-3-hydroxy-5-mesitylcyclohex-2-enone, available as GRASP.TM. from Dow Chemical USA (Midland, Mich.)
For purposes of reference in the present specification, the herbicides described in the two preceding paragraphs and other structurally related herbicidal compounds, are collectively referred to as the cyclohexanedione family of herbicides.
6 Herbicidal Aryloxyphenoxyproanoic Acids
Certain aryloxyphenoxypropanoic acids exhibit general and selective herbicidal activity against plants. In these compounds, the aryloxy group may be phenoxy, pyridinyloxy or quinoxalinyl. One such herbicidal aryloxyphenoxypropanoic acid is haloxyfop, {2-[4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid}, which is available as VERDICT.TM. Dow Chemical USA (Midland, Mich.) Another is diclofop, {(.+-.)-2-[4-(2,4-dichlorophenoxy)-phenoxy]propanoic acid}, available as HOELON.TM. from Hoechst-Roussel Agri-Vet Company (Somerville, N.J.).
Other members of this family of herbicides include fenoxyaprop, (.+-.)-2-[4-[(6-chloro-2-benzoxazolyl)oxy]phenoxy]propanoic acid; available as WHIP.TM. from Hoechst-Roussel Agri-Vet Company (Somerville, N.J.); fluazifop, (.+-.)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid; available as FUSILADE.TM. from ICI Americas (Wilmington,, Del.); fluazifop-P, (R)-2-[4-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid; available as FUSILADE 2000.TM. from ICI Americas (Wilmington, Del.); and quizalofop, (.+-.)-2-[4[(6-chloro-2-quinoxalinyl)oxy]phenoxy]propanoic acid; avialable as ASSURE.TM. from E. I. DuPont de Nemours (Wilmington, Del.).
For purposes of reference in the present specification, the herbicides referred to in the two preceding paragraphs and other structurally related herbicidal compounds, are collectively referred to as herbicidal aryloxphenoxypropanoic acids.